This invention relates to targeted antigen presentation in the immune system. In particular, this invention relates to the use of apoptotic cells to deliver antigens to dendritic cells for induction or tolerization of antigen-specific T cell responses. The apoptotic cell-mediated antigen delivery system described herein has a wide range of preventive, diagnostic and therapeutic applications.
CD8+ cytotoxic T lymphocytes (CTLs) play a critical role in immune defense against infectious agents, tumors and transplants. Class I-restricted CD8+ T cells have been implicated in the recognition and destruction of such clinically important targets as HIV-1 (1-3), Influenza A (4), malaria (5), cytomegalovirus infected cells (6), Epstein-Barr virus (7), and human melanoma cells (8-9). Therefore, establishing methods for inducing and expanding populations of antigen-specific CD8+ CTLs remains an important objective in the development of therapeutic treatments against infectious disease and cancer.
CD8+ CTLs are activated by antigens which have been processed and presented on major histocompatibility (MHC) class I molecules on the surface of specialized antigen presenting cells. A number of antigen presenting cells have been identified which activate T cells including macrophages/monocytes, B cells and bone marrow derived dendritic cells. Of these, dendritic cells are recognized as playing a pivotal role in the initiation of CD8+ CTL responses (10).
An important feature of dendritic cells is their ability to efficiently process and present antigens on MHC class I and/or class II molecules. Depending on the antigen processing pathway, dendritic cells are capable of activating distinct populations of CTLs. In the case of influenza virus, for example, it is known that the class I pathway for inducing CD8+ CTLs requires adequate delivery of infectious viral antigen into the cytoplasm, whereas the purely endocytic pathway delivers noninfectious virions for presentation only to CD4+ T helper cells (U.S. Ser. No. 08/282,966). Thus, although dendritic cells efficiently activate class I-restricted CTLs, access to the MHC class I pathway for induction of CD8+ T cells normally requires endogenous synthesis of antigen. Accordingly, it is important to identify antigen delivery systems which efficiently mediate access of exogenous antigen to the MHC class I-restricted antigen presentation pathway in order to generate antigen-specific CD8+ T cell responses.
Recently, a number of approaches have been reported for delivery of exogenous antigen to the MHC I processing pathway of dendritic cells. These methods include coupling antigens to potent adjuvants (11-15), osmotic lysis of pinosomes after pinocytic uptake of soluble antigen (16), or insertion of antigen in pH-sensitive liposomes (17). However, these prior approaches still pose a number of limitations on the development of effective therapeutic treatments. For example, Nair et al. report that dendritic cells do not efficiently internalize antigen-containing liposomes in vivo (18). Further, osmotic lysis of dendritic cell pinocytic vesicles may be difficult to perform in vivo, and may result in inefficient antigen delivery to the MHC class I processing compartment. Also, the use of powerful adjuvants may be undesirable for some clinical applications.
Pulsing dendritic cells directly with exogenous antigen using whole cells in viable or irradiated forms, membrane preparations, or antigens purified from natural sources or expressed as recombinant products has also been previously reported (WO 94/02156). These prior methods, however, do not recognize forms of cell death or the processing pathways antigens from dead or dying cells access in the dendritic cell system.
Rubartelli et al. report that dendritic cells, unlike macrophages, fail to take up opsohized particles or necrotic cells in vitro, but can efficiently engulf cells undergoing apoptotic programmed cell death (19). The mechanism of internalization of apoptotic cells by dendritic cells, however, is different than in macrophages, indicating that results in the macrophage system are not necessarily predictive of dendritic cell responses. In addition, Rubartelli et al. does not show presentation of engulfed material and therefore speculates as to the fate of such material.
This invention provides highly efficient methods for delivering exogenous antigens to dendritic cells and inducing antigen-specific T cell activation. In particular, the methods described in this invention are directed toward developing therapies for increasing patient immunity to chronic infections and tumors by 1) inducing tumor or infected cells to undergo apoptosis, 2) having the apoptotic tumor or infected cells gain access to phagocytic, maturing dendritic cells, and 3) exposing the apoptotic cell-primed dendritic cells expressing antigen of interest to T cells, in vivo or in vitro, for induction of antigen-specific T cell responses.
This invention further provides that the population of donor cells expressing said antigen can be induced to undergo apoptosis using a variety of methods including, but not limited to, viral infection, irradiation with ultraviolet light, gamma radiation, cytokine treatment, or depriving donor cells of nutrients in the cell culture medium. It is also contemplated by this invention that the dendritic cells can be exposed to a preparation of donor apoptotic cell fragments, blebs or bodies rather than whole apoptotic cells.
In another embodiment of this invention, the donor cells can be transfected, transduced or transformed to express foreign antigens prior to induction of apoptosis. A variety of such antigens may be expressed by the donor cells including, but not limited to, viral antigens, tumor antigens, toxins, microbial antigens, and autoimmune antigens.
Accordingly, this invention also provides a method of generating antigen-specific cytotoxic T lymphocytes comprising providing a population of apoptotic cells, or membrane containing fragments thereof, expressing said antigen, exposing dendritic cells to said apoptotic cells for a time sufficient to allow said antigen to be internalized and processed by the dendritic cells, and exposing T lymphocytes in vivo to said dendritic cells for a time sufficient to induce said lymphocytes to become antigen-specific T lymphocytes. This invention further contemplates induction of antigen-specific T lymphocytes in vitro.
In another embodiment of this invention apoptotic cells expressing an antigen to be presented by dendritic cells are administered to an individual in an amount and in a location so as to prime dendritic cells in vivo.
Methods of preventing and treating disease are also provided by this invention which comprises administering to an individual in need of treatment, a therapeutically effective amount of dendritic cells which have been primed by apoptotic cells.
In addition to the methods of this invention, this invention also provides dendritic cells which have been primed by apoptotic cells, and in particular, human dendritic cells which are prepared according to the methods of this invention. This invention further provides transformed apoptotic cells, or a preparation of transformed apoptotic cell fragments, and in particular, human apoptotic cells which are prepared according to the methods of this invention.
In another embodiment of this invention the apoptotic cell delivery system is reconstituted in vitro using liposomes comprising antigen and apoptotic cell proteins, factors or ligands which enhance uptake and processing of antigen in dendritic cells.
Based on the results disclosed herein, one may include in a liposome ligands for integrin receptors, including, but not limited to the xcex1vxcex25 and xcex1vxcex23 integrin receptors for enhancing uptake and processing of antigens in dendritic cells. An example of such a ligand is lactadherin. Other examples of proteins which may enhance uptake of apoptotic particles include thrombospondin. Similarly, ligands which bind CD36 may also be used. In a preferred embodiment ligands for both integrin and CD36 would be used.
Heat shock proteins (HSP) may also be included to facilitate antigen uptake by dendritic cells. For example, HSP 70 family members, especially HSP 70, and HSP 90 members, especially HSP 84 and GP 96 may be used. Certain antigens obtained from infectious sources may already be bound to HSPs. Alternatively, antigens may be complexed to HSP ex vivo.
The identification of the xcex1vxcex25 integrin receptor as mediating antigen uptake by dendritic cells provides one with a means of modulating that antigen uptake by either increasing or decreasing the activity of the xcex1vxcex25 receptor. Accordingly, this invention provides means for enhancing antigen uptake and processing by dendritic cells in vitro or in vivo, by adding an xcex1vxcex25 ligand to the dendritic cells in conjunction with the antigen to be taken up and processed. Alternatively, one could attenuate the antigen uptake activity of the dendritic cells by blocking the xcex1vxcex25 receptor, or other receptors which are involved in medrating antigen uptake by the dendritic cells.
This invention also includes pharmaceutical compositions for charging dendritic cells comprising a ligand which activates antigen uptake, such as an xcex1vxcex25 ligand, with or without an antigen to be processed and presented by dendritic cells. As used in this invention such ligands may be considered to function as immunoadjuvants.
This invention also provides methods for modulating T cell responses in vivo or in vitro to induce immunity or tolerance by controling DC maturation.
In one embodiment, T cell immunity can be induced when immature DCs are caused to mature by the addition of appropriate maturation factors after the uptake of apoptotic particles. Examples of maturation factors that can be used in this invention include monocyte conditioned medium, TNF-xcex1, IL-1xcex2, IL6, PGE2, IFN-xcex1, CD40 ligand, and necrotic cells.
In another embodiment, tolerance can be induced to self or foreign antigens by maintaining DCs in their immature state in the absence of a maturation signal.
It is a general object of this invention to provide a method of using apoptotic cells, or apoptotic cell fragments, to efficiently deliver specific antigens to dendritic cells which then process and present said processed antigens on their surface for stimulation or tolerization of cytotoxic T lymphocytes.
It is another object of this invention to generate antigen-specific cytotoxic T lymphocytes either in vivo or in vitro by using the dendritic cells generated by the methods described herein.
It is also an object of this invention to provide a method of prophylactic or therapeutic treatment for a variety of cancers, autoimmune diseases, and pathogens using the dendritic cells described herein.